PROJECT SUMMARY Intracellular protein therapies are tremendously appealing, promising treatments for many heretofore untreatable diseases, including genetic diseases and cancers. However, the safe, efficient intracellular delivery of proteins remains a challenge. Here we propose the development of an effective cytoplasmic delivery strategy for therapeutic proteins for cancer treatment: A combination of reversible chemical modification of proteins and nanocomplexation with cationic lipid-based nanoparticles. The chemical modification will inactivate the cytotoxic proteins, making them effectively nontoxic to normal cells. However, upon encountering increased level of reactive oxygen species (ROS), i.e. inside the cancer cells, the modified chemical moiety will be cleaved and the biological function of the proteins will be restored, resulting in cytotoxic action. Simultaneously, chemical modification reduces the surface charge of proteins, leading to more efficient nanocomplexation and delivery by bioreducible cationic lipid-like molecules. This protein delivery platform may thus serve as an efficient tumor- specific targeting system for cancer treatment. To accomplish the objective of this application, we propose the following three Specific Aims: Specific Aim 1: ROS-responsive modification of saporin and its characterization. To develop an approach for chemical modification of saporin, characterize the modification, and investigate the cleavage of the modified moieties at elevated ROS levels. Specific Aim 2: Intracellular delivery of saporin-NBC using synthetic lipids. To study a new class of synthetic lipid-based nanoparticles for intracellular saporin-NBC delivery in both cancerous and non-cancerous cells, and to study the synergistic effect of intracellular delivery of saporin-NBC and ROS-inducing anticancer drugs in suppressing triple-negative breast cancer cells. Specific Aim 3: Studying the in vivo saporin-NBC delivery in mouse breast cancer model. To study the therapeutic efficacy of bioreducible lipids in delivering proteins to inhibit tumor growth in murine breast cancer mouse models.